Bovine oocytes in secondary follicles grow in medium containing bovine plasma after vitrification

There has been no culture system that supports the growth of bovine oocytes for more than 2 weeks. In the present study, bovine secondary follicles were cultured for 4 weeks, and the effects of supplemented protein components and FSH in the culture medium on the growth of the oocytes were examined. The effect of vitrification of secondary follicles on the subsequent oocyte growth was also examined. Secondary follicles (150 to 200 μm in diameter) containing growing oocytes (approximately 60 μm in diameter) were dissected from ovaries and cultured in a medium supplemented with FSH (0, 25 or 50 ng/ml) and one of the following four kinds of protein components: bovine serum albumin (BSA), bovine plasma (BPL), fetal calf serum (FCS) and bovine follicular fluid (BFF). In BSA- and BPL-supplemented media with 0 or 25 ng/ml FSH, more than 50% of follicles showed no degenerative signs during culture, and oocytes significantly increased in size after 4 weeks (P<0.05). Higher percentages of granulosa cell-enclosed oocytes were recovered from the follicles cultured in BPL-supplemented media with 0 and 25 ng/ml FSH, and the oocytes grew to 90 μm or more in diameter. In FCS- and BFF-supplemented media, FSH increased the numbers of degenerating follicles. Next, vitrified-warmed secondary follicles were cultured in BPL-supplemented medium. One third of the follicles showed no degenerative signs, and the oocytes increased in diameter to 88.8 ± 3.1 μm after 4 weeks of culture. These results suggest that a BPL-supplemented medium supports oocyte growth in bovine secondary follicles for 4 weeks, even after vitrification and warming of the follicles.     

Mycoplasma ovis detected in free-living Japanese serows, Capricornis crispus

Nineteen blood samples collected from free-ranging wild Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the hemoplasma infection by real-time PCR targeting the 16S rRNA gene. Five (26.3%) out of the 19 samples were positive in real-time PCR with an average melting temperature at 85.18 °C. The positive samples in the real-time PCR were reconfirmed by conventional PCR, and one of them was successful for direct DNA sequencing. The nucleotide sequence of the 16S rRNA gene of the representative stain was identical to that of Mycoplasma ovis. This was the first demonstration of hemotropic mycoplasma infections among the free-living Japanese serows in Japan.     

Differential kinetic activities of glycerol kinase among African trypanosome species: phylogenetic and therapeutic implications

African trypanosome species are causative agents for sleeping sickness in humans and nagana disease in cattle. Trypanosoma brucei can generate ATP via a reverse reaction with glycerol kinase (GK) when alternative oxidase (AOX) is inhibited; thus, GK is considered to be a crucial target for chemotherapy combined with AOX. However, the energy metabolism systems of African trypanosome species other than T. brucei are poorly understood. Thus, GK genes were surveyed from genome databases and cloned by PCR from T. vivax and T. congolense. Then, recombinant GK proteins (rGK) of T. vivax, T. congolense and T. brucei were expressed and purified. Kinetic analysis of these rGK proteins revealed that the K(m) values of T. congolense rGK for ADP and G-3-P substrates were lower than those of T. vivax and T. brucei. The expression level of GK molecules was highest in T. congolense cells and lowest in T. vivax cells. Based on these results, effective combination dosages of ascofuranone, a specific inhibitor of AOX, and glycerol, an inhibitor of the GK reverse reaction, were determined by using in vitro-cultured trypanosome cells.     

Follicle-stimulating hormone (FSH) stimulates the expression of Pin1, a peptidyl-prolyl isomerase, in the bovine granulosa cells

A peptidyl-prolyl isomerase, Pin 1, has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo. However, the involvement of Pin 1 during follicular development is not well understood. The aim of this study was first to investigate the expression of Pin 1 mRNA in the granulosa and theca cells of the follicle at different developmental stages of follicles in the bovine ovary, and second, to examine the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of Pin 1 in the cultured bovine granulosa cells. Follicles were classified into four groups based on the diameter (dominant follicles >8.5mm in diameter, subordinate follicles <8.5mm in diameter) and the relative levels of E2 and progesterone (P4) (E2:P4>1, estrogen active; E2:P4<1, estrogen inactive): i.e. preovulatory dominant follicles (POFs); E2 active dominant follicles (EADs); E2 inactive dominant follicles (EIDs); small follicles (SFs). The expression of the Pin 1 gene was significantly increased in the granulosa cells of EADs as compared with those of other follicles, whereas its expression in theca cells did not differ among follicles at different developmental stages. The concentration of 5 ng/ml FSH alone and the combination of 1 ng/ml E2 and 5 ng/ml FSH stimulated the expression of the Pin 1 gene in bovine granulosa cells. Our data provide the first evidence that Pin 1 expression in the granulosa cells but not the theca cells changes during follicular development, and that FSH stimulate the expression of the Pin 1 gene. These results suggest that Pin 1 regulates the timing of cell proliferation and may act as an intracellular signal responder in the granulosa cells during bovine follicle development.     

Effects of enalapril in cats with pressure overload-induced left ventricular hypertrophy

In order to evaluate the effect of enalapril on haemodynamics and renal function in a pressure overload model, we prepared eight feline models of left ventricular hypertrophy (LVH) by banding of the aortic arch. The LVH cats were assigned to the placebo group or the enalapril group (0.5 mg/kg, PO, sid) 3 months following surgery, and each received its respective drug for 4 weeks. Each week, blood pressure, angiotensin converting enzyme (ACE) activity in blood, and creatinine clearance were measured, and complete blood count (CBC), biochemical examination of the blood, echocardiography, and chest radiography were carried out. The interventricular septum thickness (IVSd, IVSs), fractional shortening (FS), and ejection fraction (EF) increased significantly in the LVH cats following surgery (P<0.05). There was no significant difference between the placebo group and the enalapril group with respect to general physical parameters, CBC, biochemical parameters and renal function. In the enalapril group, systolic arterial pressure, mean arterial pressure, and ACE activity in blood decreased significantly following administration (P<0.05). In addition, the left ventricular free wall thickness in diastole and IVSd decreased significantly following administration (P<0.05). These results suggest that, in a pressure overload model, enalapril (0.5 mg/kg, sid) inhibits cardiac hypertrophy, reduces blood pressure, and does not adversely affect renal function.     

Evaluation of the distance between Schwalbe’s line and the anterior lens capsule as a parameter for the correction of ultrasound biomicroscopic values of the canine iridocorneal angle

Objective To determine whether the distance between Schwalbe’s line (the peripheral termination of Descemet’s membrane, i.e., the borderline between the cornea and sclera) and the anterior lens capsule (SLD) is an applicable parameter for correcting raw ultrasound biomicroscopic values of the canine iridocorneal angle (ICA) and to establish a comparative system for the ICA values in different canine breeds with varied body sizes/weights. Animal studied The dogs were divided into four groups based on body weight (BW): <4 kg, 4–8 kg, 8–20 kg, and >20 kg, and 180 normotensive eyes were studied. Procedures The ICA microstructure was examined by ultrasound biomicroscopy (UBM) using 40‐MHz probes in dogs with or without anesthesia/sedation. Linear regression analysis and correlation coefficients were evaluated between SLD or SLD² and UBM measurements; subsequently, noncorrected and SLD‐corrected UBM values were statistically assessed. Results Significant linear correlations were detected between SLD and the ciliary cleft width, the minimum distance between the angle recess and the scleral venous plexus, and the scleral thickness. Positive linear correlations were also demonstrated between SLD² and the ciliary cleft area as well as the scleral venous plexus area. Raw UBM measurements were corrected by using a ratio with SLD on the distance or SLD² on the area. Although noncorrected UBM measurements increased with canine BW, SLD‐corrected UBM values remained similar with no significant statistical differences in any of the dogs. Conclusions Correction with SLD would be clinically useful for comparing UBM measurements of the ICA in dogs with different body sizes/weights.     

Two Eimeria species isolated from wild Japanese rock ptarmigans (Lagopus mutus japonicus) in Japan

Fecal samples were collected from 64 Japanese rock ptarmigans (Lagopus mutus japonicus) at 13 locations in the alpine zone of Japan and examined for internal parasites. We found 2 morphologically different types of eimerian oocysts. Based on morphological characteristics, one was identified as Eimeria uekii, which had already been found from Japanese rock ptarmigans in 1981, and the other was likely a new type of Eimeria. The new type of eimerian oocysts required a longer sporulation time than E. uekii. The prevalence of both eimerian oocysts in Japanese rock ptarmigans was 62.5%, while that of E. uekii was 60.9% and the new type 29.7%. Mixed infection of both types was found in 28.1%.     

Expression of neural markers on bone marrow-derived canine mesenchymal stem cells

OBJECTIVE: To evaluate cell surface markers of bone marrow-derived canine mesenchymal stem cells (MSCs) by use of flow cytometric analysis and determine whether canine MSCs express proteins specific to neuronal and glial cells. SAMPLE POPULATION: Bone marrow aspirates collected from iliac crests of 5 cadavers of young adult dogs. PROCEDURES: Flow cytometric analysis was performed to evaluate cell surface markers and homogeneity of third-passage MSCs. Neural differentiation of canine MSCs was induced by use of dibutyryl cAMP and methyl-isobutylxanthine. Expressions of neuronal (beta III-tubulin) and glial (glial fibrillary acidic protein [GFAP] and myelin basic protein) proteins were evaluated by use of immunocytochemical and western blot analyses before and after neural differentiation. RESULTS: Third-passage canine MSCs appeared morphologically homogeneous and shared phenotypic characteristics with human and rodent MSCs. Immunocytochemical and western blot analyses revealed that canine MSCs constitutively expressed beta III-tubulin and GFAP. After induction of neural differentiation, increased expression of GFAP was found in all samples, whereas such change was inconsistent in beta III-tubulin expression. Myelin basic protein remained undetectable on canine MSCs for these culture conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Canine bone marrow-derived mononuclear cells yielded an apparently homogeneous population of MSCs after expansion in culture. Expanded canine MSCs constitutively expressed neuron or astrocyte specific proteins. Furthermore, increases of intracellular cAMP concentrations induced increased expression of GFAP on canine MSCs, which suggests that these cells may have the capacity to respond to external signals. Canine MSCs may hold therapeutic potential for treatment of dogs with neurologic disorders.     

In vitro growth of bovine oocytes in oocyte-cumulus cell complexes and the effect of follicle stimulating hormone on the growth of oocytes

Several successful in vitro culture experiments have used oocyte-cumulus cell-mural granulosa cell complexes (OCGCs) from early antral follicles (0.5–0.7 mm) for the growth of bovine oocytes. However, in studies related to in vitro oocyte maturation and in vitro embryo production, oocyte-cumulus cell complexes (OCCs) that have no mural granulosa cells have been widely used instead of OCGCs. The purpose of this study was to determine whether cumulus cells alone support oocyte growth. First, OCCs and OCGCs were cultured in vitro for 14 days to compare the integrity of the complexes as well as antrum formation. After 14 days, the diameter and meiotic competence of oocytes in OCCs and OCGCs were examined. Oocytes in OCCs grew fully and acquired meiotic competence similar to OCGCs, whereas antrum formation occurred later in OCCs as compared to OCGCs. Subsequently, the effects of follicle stimulating hormone (FSH) on in vitro growth of OCCs were examined for 14 days. When FSH was added to the culture medium, OCCs formed antrum-like structures one day earlier than those cultured without FSH. Oocytes cultured with 1 mIU/ml FSH grew fully and acquired meiotic competence. In contrast, when oocytes were cultured in media containing high concentrations of FSH, some of the OCCs collapsed and the number of degenerated oocytes increased. In conclusion, bovine oocytes in OCCs grow and acquire meiotic competence similar to OCGCs and, 1 mIU/ml FSH supports the development of OCCs and oocyte growth as observed in our culture system.